Isolating specific target nucleic acids from a sample is an important step for many medical diagnostic assays. For example, certain mutations and methylation states of particular genes are correlated, associated, and/or predictive of disease. DNA harboring these genes can be recovered from a sample and tested for the presence of the particular mutations and methylation states. While assaying an individual gene is informative, a more sensitive and specific diagnostic assay targets suites of genes comprising many members. Such an assay also provides additional information relating to the origin and stage of disease.
In practice, assaying such a multi-gene panel requires isolating and assaying several genetic targets from a sample. For many detection methods, detecting rare mutations or methylation events in a single gene requires isolating and testing a large quantity of DNA to obtain sufficient copies of the gene to allow detection of the rare event. This problem is compounded when assaying a panel of genes, each of which must be present in a large quantity for a robust diagnostic test. Thus, to detect rare mutations and methylation events in multiple genes, the isolated DNA must be highly concentrated and comprise a substantial portion of the detection assay. This requirement imposes many problems, however. For example, preparing such quantities and concentrations of DNA requires a large sample as input (i.e., having a mass of many grams), and thus requires a method that can prepare DNA from a large sample. In addition, assay inhibitors are often isolated and concentrated with the DNA preparation. Consequently, conventional assays that require concentrated DNA also often introduce inhibitory amounts of inhibitors into the assay. Moreover, if all targets of the panel are extracted and assayed simultaneously, the sensitivity of the assay is compromised because each gene of the panel composes a smaller fraction of the preparation as more targets are isolated. Also, if all members of the panel are extracted together and thus present in the same solution, the sensitivity of an assay directed to detecting any single particular target is compromised by interference from the non-target DNA molecules.
In addition, if a particular diagnostic target is present in a sample, it will be present in a small amount, thus providing a challenge for methods designed to detect it. For example, analyses of DNA from stool samples are complicated by the fact that large amounts of DNA from other sources are present. For example, bacteria compose approximately 60% of the dry mass of feces and the remainder is largely the remains of plant and animal matter ingested as food by the subject. As such, the human subject's cells, and only those that slough off the lining of the digestive tract, are a very small fraction of the stool. Furthermore, in assays to detect gene modifications indicative of colon cancer, if a cancerous tumor is present in the colon, then cells derived from that tumor would compose only a small fraction of the human subject's gut cells that slough off the digestive tract lining. Consequently, cancer cells (and the DNAs they contain) make up a minimal amount of the stool mass.
Conventional methods and kits for isolating DNA from samples typically prepare total DNA (i.e., non-specifically) from the sample. Consequently, total DNA isolated from a stool sample comprises DNA from the gut-resident bacteria (and any viruses, eukaryotes, and archaea present) along with DNA from the subject sampled. Moreover, conventional methods and kits are primarily designed to prepare DNA from small samples, e.g., samples having masses of less than 1 gram, e.g., 50 to 200 milligrams. Consequently, conventional methods are not applicable to high-sensitivity and high-specificity multi-gene panel analyses because they cannot prepare sufficient amounts of highly concentrated, inhibitor-free DNA from large samples such as a stool sample. Assays using DNA prepared with conventional methods will not provide a sample that can be assayed with the required sensitivity threshold for detecting rare mutation or methylation events. Using a conventional method or kit to provide the starting quantities needed to attain such sensitivity requires multiple DNA extractions (e.g., the use of multiple kits) from multiple samples in addition to extra purification steps to remove inhibitors. Therefore, what is needed is a method of preparing concentrated, inhibitor-free DNA from a sample for each member of a gene panel for use in diagnostic assays.